We are continuing to develop new methods for analysis of important neurobiologic properties using a variety of cell culture systems, and to apply these analytical techniques to fundamental neurobiologic problems. Our aim is an understanding of the regulatory phenomena which occur with enzyme induction. We have continued to evaluate the inductive effect on spinal cord (SC) cell enzymes of co-culture with muscle cells and of muscle conditioned medium. We have determined that the considerable activity of the enzyme glutamate decarboxylase (GAD) in pineal glands is not regulated by norepinephrine, diurnal cycle, superior cervical ganglionectomy or ablation of the habenular nucleus. We have developed a new radiometric assay for GAD activity which solves the many difficulties found in other assays. By growing primary fetal rat brain cells for 2 weeks in conditioned medium from iodo-deoxyuridine- treated mouse neuroblastoma (N-18) cells, and then maintaining the cells with normal medium for about 2 months, we obtained transformed cells with rat karyotype which have been colony-cloned and are being characterized. We have recently been successful in obtaining cell cultures of rat pineal cells in mixed culture with some other cell types. We feel that our continuing interest in factors which regulate neurobiologic phenomena will lead to findings which may help to explain deficits in degenerative and developmental states. Culture systems have promise for a dissection of the molecular events which surround formation of synaptic contacts and functioning synapses. Such information will be of value in understanding nerve regeneration and abnormalities of nervous system development.